Carbohydrate based anti-bacterials

ABSTRACT

A method of inhibiting bacterial growth by contacting a bacteria with at least one disaccharide compound of General Formula I,

FIELD OF THE INVENTION

This application is the US national phase of international applicationPCT/AU2003/001377 filed 16 Oct. 2003, which designated the U.S. andclaims benefit of AU 2002952121, dated 17 Oct. 2002, the entire contentsof each of which are hereby incorporated by reference.

The invention relates to disaccharide compositions that haveantibacterial properties.

BACKGROUND OF THE INVENTION

Bacteria have a great ability to generate resistance to drugs throughlateral gene transfer, mutation of enzymes, or by expressing enzymeswhich actively pump out the drug or break it down. Over the past 10years resistance to existing drugs has become a significant problem inmany countries. No new antibacterial drugs have been developed over thepast 15 years. Vancomycin is currently the drug of last resort to combatthe multidrug resistant Gram-positive bacteria In many placesvancomycin-resistant Staphylococcus aureus and Enterococci (VRE) havebeen discovered. There is thus a desperate need for a new antibacterialdrug to replace the drug of last resort.

There are a host of cytoplasmic targets for the development of newantibacterials, such as gyrase inhibitors, protein synthesis inhibitors,muramyl cascade inhibitors and many more. The major hurdle in designingsuch drugs is that in addition to enzyme based activity these drugs needto cross the bacterial cell wall to exert their antibacterial effect. Onthe other hand, enzymes involved in the stage III synthesis of thebacterial cell wall exist on the cell wall exterior, and therefore drugsinhibiting these enzymes can exert their bactericidal or bacteriostaticeffect without having to cross the cell wall. Penicillin, cephalosporinand vancomycin are drugs that act on the transpeptidase enzymes whichcontrol the final steps in the peptidoglycan biosynthesis. Moenomycin isknown to act on the transglycosylase enzymes, which are similarlyinvolved in the polymerization of disaccharide precursors. Moenomycindisplays very high potency at MIC level, and is used in animal feed as agrowth promoter.

Moenomycin is a lipid-linked pentasaccharide. Through extensive SARexperiments it was realised that smaller fragments of moenomycin werecapable of exerting antibacterial activity. Trisaccharide fragments ofmoenomycin still display antibacterial activity, but are notsufficiently stable to be useful drugs. On the basis of this, Sofia andcoworkers discovered a new series of disaccharides, carrying aromaticsubstituents in well defined positions around the disaccharide, whichdisplayed significant MIC activity [WO0064915 and WO9926596].

A further class of disaccharide molecules, based on a sub-structure ofvancomycin was shown to have antibacterial activity against vancomycinresistant bacteria. This class of molecules was subsequentlydemonstrated to contain transglycosylase inhibitors, and were nottranspeptidase inhibitors as is vancomycin itself [WO9853813].

SUMMARY OF THE INVENTION

The present invention is directed to antibacterial compositions and isespecially directed to a method of reducing bacterial growth bycontacting bacteria with particular disaccharide like moieties.

The present invention may also be directed to an antibacterialpharmaceutical composition comprising a pharmaceutically acceptablecarrier and a therapeutically effective amount of at least oneparticular disaccharide like moiety.

The present invention may also be directed to a method of screening suchcompounds for anti-bacterial activity by contacting the compounds with aGram-positive or Gram-negative bacteria and monitoring the growth orgrowth inhibition of the bacteria.

In a first aspect, the invention provides a method of inhibitingbacterial growth by contacting a bacteria with at least one disaccharidecompound of General Formula I,

Wherein the pyranose rings may be of any configuration,

-   T is either R or —XR, where X is defined as oxygen, sulphur,    NHC(O)—, and wherein R is selected from the non-limiting set    comprised of H, or an alkyl, alkenyl, alkynyl, heteroalkyl aryl,    heteroaryl, arylalkyl or heteroarylalkyl of 1 to 20 atoms which is    optionally substituted, and can be branched or linear. Typical    substituents include but are not limited to OH, NO, NO₂, NH₂, N₃,    halogen, CF₃, CHF₂, CH₂F, nitrile, alkoxy, aryloxy, amidine,    guanidiniums, carboxylic acid, carboxylic acid ester, carboxylic    acid amide, aryl cycloalkyl, heteroalkyl, heteroaryl, aminoalkyl    aminodialkyl, aminotrialkyl, aminoacyl, carbonyl, substituted or    unsubstituted imine, sulfate, sulfonamide, phosphate, phosphoramide,    hydrazide, hydroxamate, hydroxamic acid, heteroaryloxy, aminoalkyl    aminoaryl aminoheteroaryl, thioalkyl thioaryl or thioheteroaryl,    which may optionally be further substituted,-   U and Z independently selected from OR, NHR, NR(R) (where R may be    the same or different), or the following non-limiting set,

-   R¹ and R² are independently selected from H, CH₃, CH₂X, and C(O)NH,-   R³ and R⁴ are independently selected from H, OH, OR, NHCOR, and-   W is independently selected from OR^(L), NHR^(L), NR^(L)R, or the    following the following non-limiting set,

Wherein R^(L) is a substituted or unsubstituted, linear or branched,saturated or unsaturated C3 to C55 alkyl, heteroalkyl, arylalkyl,alkylaryl chain. Substituents may include but are not limited to acidicgroups such as carboxylic acids, sulfonic acids, phosphoric acids,tetrazoles, or other carboxylic acid mimetics or basic groups such asamines, guanidines, amidines, imidazoles or other amine mimetics.

In a further aspect, the invention provides a method of inhibitingbacterial growth by contacting a bacteria with at least one disaccharidecompound of General Formula II,

Wherein the disaccharide linkage is alpha or beta,

-   A is defined as hydrogen, OR or SR, and-   R, U, W, Z and R⁴ are defined as in General Formula I.    In a more preferred aspect, the invention provides a method of    inhibiting bacterial growth by contacting a bacteria with at least    one disaccharide compound of General Formula III,

Wherein A is defined as in General Formula I, and

-   U, Z, R^(L) and R⁴ are defined as in General Formula I.

The bacterial may be Gram-positive or Gram-negative bacteria. Thebacteria may comprise an E-coli bacteria, a Staphylococci Bacteria suchas Staphylococcus aureus, or other bacteria such as Micrococcus luteus(ATCC272), Staphylococcus aureus (ATCC29213), Staphylococcus aureus(ATCC43300) MRSA, Enterococcus faecalis (ATCC29212), Enterococcusfaecalis (ATCC51299) Vancomycin resistant and Streptococcus pyogenes(ATCC8668).

The method may comprise administering an effective amount of a compoundof the first aspect, to a subject in need of such treatment. The subjectmay be a human, or may be a domestic, companion or zoo animal.

In another form, the invention may reside in an antibacterialcomposition comprising at least one compound as described above. Thecomposition may comprise a pharmaceutical composition.

The compounds of the invention may be mixed with a pharmaceuticalacceptable carrier, adjuvant, or vehicle which may comprise a-toxiccarrier, adjuvant, or vehicle that may be administered to a patient,together with a compound of this invention, and which does not destroythe pharmacological activity thereof.

The pharmaceutical derivative may comprise a salt, ester, sit of anester or other derivative of a compound of this invention which, uponadministration to a recipient, is capable of providing, either directlyor indirectly, a compound of this invention, although no limitation ismeant thereby.

Compounds of the invention may be administered orally such as by meansof a tabled, powder, liquid, emulsion, dispersion and the like; byinhalation; topically such as by means of a cream, ointment, salve etc;and as a suppository, although no limitation is meant thereby.

Methods and pharmaceutical carriers for preparation of pharmaceuticalcompositions are well known in the art, as set out in textbooks such asRemington's Pharmaceutical Sciences, 19th Edition, Mack PublishingCompany, Easton, Pa., USA.

It will be clearly understood that, if a prior art publication isreferred to herein, this reference does not constitute an admission thatthe publication forms part of the common general-knowledge in the art inAustralia or in any other country.

Best Mode

MIC Testing:

The broth microdilution format of the National Committee for ClinicalLaboratory Standards (NCCLS) approved standard for susceptibility testsas outlined in M7-A4 “methods for dillution Antimicrobial susceptibilitytests for bacteria that grow aerobically; approved standard—fifthedition”, January 2000 was utilized for minimum inhibitory concentrationtesting in Mueller-Hinton broth. The broth for Streptococcus pyogenestesting was supplemented with 2% laked horse blood. A positive result ininitial testing was determined by complete inhibition of macroscopicbacterial growth at a concentration of 128 micrograms per mL afterincubation for 16 to 24 hours at 37 degrees C. In the case ofMicrococcus luteus, incubation was at 30 degrees C.

EXAMPLE 1

Comp. No. R1 R2 Mass R_(f) SA24 SA48 EC24 1 A5 A9 679 4.62 + n.d. −In all examples, + indicates an MIC value of less than 128 microgramsper mL, − indicates an MIC of greater then 128 micrograms per mL andn.d. indicates not determined.Bacterial Types Are:

-   SA24 S. aureus after 24 hours exposure-   SA48 S. aureus after 48 hours exposure-   EC24 E. coli after 24 hours exposure

EXAMPLE 2

Comp. No. n X Y R2 R3 MS R_(f) SA24 SA48 EC24 2 1 A1 A10 A11 A7 875n.d + + − 3 1 A1 A10 A4 A9 831 n.d + + − 4 0 A1 A10 A12 A9 800 5.1  +n.d − 5 0 A1 A10 A5 A7 862 4.92 + + − 6 0 A1 A10 A5 A9 851 5.36 + n.dn.d. 7 1 A10 A1 A5 A7 876 5.01 + + −

EXAMPLE 3

Comp. No. R2 R3 MW R_(f) SA24 SA48 EC24 8 A5 A7 824 4.72 + + − 9 A5 A9813 5.56 + n.d. n.d.

EXAMPLE 4

Comp. No. X Y R2 R3 MW Rf SA24 SA48 EC24 10 A1 A10 A12 A7 875 n.d. + + −11 A1 A10 A4 A9 831 5.18 + + + 12 A1 A10 A4 A7 843 4.65 + + + 13 A1 A10A4 A1 663 3.2 + n.d. n.d. 14 A1 A10 A5 A9 864 5.27 + + − 15 A1 A10 A19A9 863 4.85 + + + 16 A1 A10 A19 A7 875 4.23 + + + 19 A1 A10 A19 A25 8494.8 + + + 20 A1 A10 A19 A22 861 3.49 + + + 21 A1 A10 A19 A16 8893.57 + + + 22 A1 A10 A19 A23 930 5.1 + + + 23 A1 A10 A19 A26 8313.56 + + + 24 A1 A10 A19 A27 899 4.22 + + + 25 A1 A10 A19 A28 9043.3 + + + 26 A1 A10 A19 A29 918 3.5 + + + 27 A14 A1 A2 A9 800 5.01 +n.d. + 28 A14 A1 A3 A9 762 4.81 + n.d. + 29 A14 A1 A12 A9 764 4.92 +n.d. + 30 A14 A1 A4 A9 782 4.9 + n.d. + 31 A14 A1 A15 A9 765 5.4 + n.d.+

EXAMPLE 5

Comp. No. X Y R2 R3 MW Rf SA24 SA48 EC24 36 A10 A1 A17 A7 919 n.d. + +n.d. 37 A10 A1 A5 A7 898 4.99 + + − 38 A1 A13 A2 A9 818 5.56 + n.d. n.d.39 A1 A13 A5 A7 844 4.72 + + − 40 A1 A13 A5 A9 833 5.63 + n.d. −

EXAMPLE 6

Comp. R1 R2 R3 MW Rf SA24 SA48 EC24 42 A20 A20 A8 963 n.d. + n.d. n.d.43 A5 A1 A7 690 n.d. + n.d. n.d. 44 A5 A3 A7 824 n.d. + + n.d. 45 A5 A3A1 644 3.82 + n.d. 46 A5 A21 A7 822 4.73 n.d. + − 47 A5 A21 A1 6423.39 + n.d. n.d. 48 A5 A17 A7 898 n.d. + + − 49 A5 A4 A7 844 4.9 n.d. +− 50 A5 A4 A1 664 3.8 + n.d. n.d. 51 A5 A4 A9 n.d. n.d. n.d. n.d. 52 A5A44 A7 823 3.98 n.d. + − 55 A5 A5 A25 851 5.47 + + n.d. 56 A5 A5 C₁₀H₂₁837 5.38 + + n.d. 57 A5 A5 A39 857 4.9 + + n.d. 58 A5 A5 A40 8615.01 + + n.d. 59 A5 A5 A22 n.d. + + − 60 A5 A5 bis-pentyl 837 4.9 + +n.d. 61 A5 A5 A32 851 5.56 + + n.d. 62 A5 A5 A31 837 5.08 + + n.d. 63 A5A5 A30 823 5.1 + + n.d. 64 A5 A5 A33 929 5.82 + + n.d. 65 A5 A5 A34 9425.17 + + n.d. 66 A5 A5 A41 938 4.81 − n.d. n.d. 67 A5 A5 A42 952 4.89 −n.d. n.d. 68 A5 A5 A32 901 5.36 − n.d. n.d. 69 A5 A5 A36 901 5.45 + n.d.n.d. 70 A5 A5 A37 795 4.62 − n.d. n.d. 71 A5 A5 A46 880 4.62 − n.d. n.d.72 A5 A5 A47 880 4.81 − n.d. n.d. 73 A5 A5 A6 893 5.1 + n.d. n.d. 74 A5A5 A7 877 4.99 + n.d. n.d. 75 A5 A5 A23 932 5.63 + n.d. n.d. 76 A5 A5 A8893 6.09 + n.d. n.d. 77 A5 A5 A9 865 5.63 + + − 78 A5 A3 A9 813 5.45 +n.d. n.d. 79 A5 A4 A9 833 5.73 + n.d. n.d. 80 A18 A4 A9 744 n.d. + n.d.n.d.The following compounds were tested against additional organisims withthe following results.

-   -   1. Micrococcus luteus (ATCC272)    -   2. Staphylococcus aureus (ATCC29213)    -   3. Staphylococcus aureus (ATCC43300) MRSA    -   4. Enterococcus faecalis (ATCC29212)    -   5. Enterococcus faecalis (ATCCS 1299) Vancomycin resistant    -   6. Streptococcus pyrogenes (ATCC8668)

Compound 1 2 3 4 5 6 76 + + + + + + 42 + + + + + + 75 + + + + + + 68 + +− + − + 65 + − − + − + 69 + + + + − + 70 + − − + − + 73 + + + + + +74 + + + + + + 66 − − − − − + 67 + + + + + + 77 + + + + + +51 + + + + + + 56 + + + + + +

TABLE 1 Side Arms

Throughout the specification and the claims unless the context requiresotherwise, the term “comprise”, or variations such as “comprises” or“comprising”, will be understood to apply the inclusion of the statedinteger or group of integers but not the exclusion of any other integeror group of integers.

It should be appreciated that various other changes and modificationscan be made to any embodiment described without departing from thespirit and scope of the invention.

1. A method of inhibiting bacterial growth comprising contacting abacteria with at least one disaccharide compound of

wherein U and Z are independently selected from the group consisting of—OR, —NHR, —NR(R),

wherein R may be the same or different, R is a moiety of not more than20 carbon atoms independently selected from the group consisting of;alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, arylalkyl andheteroarylalkyl; W is independently selected from the group consistingof —OR^(L), —NHR^(L), —NR^(L)R,

wherein R^(L) is a substituted or unsubstituted, linear or branchedmoiety of between 3 and 55 carbon atoms selected from the groupconsisting of: alkyl, heteroalkyl, arylalkyl, and alkylaryl chain. 2.The method of claim 1, wherein R^(L) is substituted by a moiety selectedfrom the group consisting of: carboxylic acids, sulfonic acids,phosphoric acids, tetrazoles, amines, guanidiniums, amidines,imidazoles, and oxazoles.
 3. The method of claim 1, wherein one or moreR groups is substituted by a moiety selected from the group consistingof: —OH, —NO, —NO₂, —NH₂, —N₃, halogen, —CF₃, —CHF₂, —CH₂F, nitrile,alkoxy, aryloxy, amidine, guanidiniums, carboxylic acid, carboxylic acidester, carboxylic acid amide, aryl, cycloalkyl, heteroalkyl, heteroaryl,aminoalkyl, aminodialkyl, aminotrialkyl, aminoacyl, carbonyl,substituted or unsubstituted imine, sulfate, sulfonamide, phosphate,phosphoramide, hydrazide, hydroxamate, hydroxamic acid, heteroaryloxy,carbamoyl, aminoaryl, aminoheteroaryl, thioalkyl, thioaryl andthioheteroaryl.
 4. The method of claim 1, wherein the compound is of


5. The method of claim 1, wherein the bacteria is a Gram+bacteria. 6.The method of claim 1, wherein the bacteria is a Gram−bacteria.
 7. Themethod of claim 1, wherein the bacteria is selected from the groupconsisting of an Escherichia coli (E. coli), Micrococcus luteus,Staphylococcus aureus, Methicillin-resistant Staphylococcus aurcus(MRSA), Enterococcus faecalis, Enterococcus faecalis Vancomycinresistant and Streptococcus pyogenes.
 8. The method of claim 1, whereinthe bacteria is Staphylococcus aureus and the compound is


9. The method of claim 1, wherein the bacteria is Staphylococcus aureusand the compound is

wherein: n X Y R2 R3 1 H CF₃

1 H CF₃

0 H CF₃

0 H CF₃

0 H CF₃

1 CF₃ H


10. The method of claim 1, wherein the bacteria is Staphylococcus aureusand the compound is

wherein: R2 R3


11. The method of claim 1, wherein the bacteria is Staphylococcus aureusand the compound is

wherein: X Y R2 R3 H CF₃

H CF₃

H CF₃

H CF₃

H H CF₃

H CF₃

H CF₃

H CF₃

H CF₃

H CF₃

H CF₃

H CF₃

H CF₃

H CF₃

H CF₃

F H

F H

F H

F H

F H


12. The method of claim 1, wherein the bacteria is Staphylococcus aureusand the compound is

wherein: X Y R2 R3 CF₃ H

CF₃ H

H CH₃

H CH₃

H CH₃


13. The method of claim 1, wherein the bacteria is Staphylococcus aureusand the compound is selected from the group consisting of:

wherein R1 R2 R3

H

H

H

H

n-decyl

and


14. The method of claim 1, wherein the bacteria is E. coli and thecompound is

wherein: X Y R2 R3 H CF₃

H CF₃

H CF₃

H CF₃

H CF₃

H CF₃

H CF₃

H CF₃

H CF₃

H CF₃

H CF₃

H CF₃

H CF₃

H CF₃

H CF₃

H CF₃

H CF₃


15. The method of claim 1, wherein the compound is

wherein: Compound R1 R2 R3 42

51

56

n-decyl 65

67

68

69

70

73

74

75

76

77

and the bacteria is Micrococcus luteus.
 16. The method of claim 1,wherein the compound is

wherein: Compound R1 R2 R3 42

51

56

n-decyl 65

67

68

69

70

73

74

75

76

77

and the bacteria is Staphylococcus aureus.
 17. The method of claim 1,wherein the compound is

wherein: Compound R1 R2 R3 42

51

56

n-decyl 67

68

69

73

74

75

76

77

and wherein the bacteria is Staphylococcus aureus MRSA.
 18. The methodof claim 1, wherein the compound is

wherein: Compound R1 R2 R3 42

51

56

n-decyl 65

67

68

69

70

73

74

75

76

77

and the bacteria is Enterococcus faecalis.
 19. The method of claim 1,wherein the compound is

wherein Compound R1 R2 R3 42

51

56

n-decyl 65

67

68

69

70

73

74

75

76

77

and wherein the bacteria is Enterococcus faecalis Vancomycin resistant.20. The method of claim 1, wherein the compound is

wherein: Compound R1 R2 R3 42

51

56

n-decyl 65

67

68

69

70

73

74

75

76

77

and the bacteria is Streptococcus pyogenes.
 21. A method of inhibiting abacterial infection in a mammal comprising administering to said mammalan effective amount of a compound of General Formula (I),

wherein U and Z are independently selected from the group consisting of:—OR, —NHR, and —NR(R),

wherein R may be the same or different, R is a moiety of not more than20 carbon atoms independently selected from the group consisting ofalkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, arylalkyl andheteroarylalkyl; W is independently selected from the group consistingof —OR^(L), —NHR^(L), —NR^(L)R,

wherein R^(L) is a substituted or unsubstituted, linear or branchedmoiety of between 3 and 55 carbon atoms selected from the groupconsisting of: alkyl, heteroalkyl, arylalkyl, and alkylaryl chain. 22.The method of claim 1, wherein the bacterium is a resistant orsusceptible strain of a Micrococcus, Streptococcus, Enterococcus orStaphylococcus.